Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
  • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/815—Enzyme separation or purification by sorption

Definitions

• the present invention is directed to a new process for the production of pure urokinase.
• Urokinase is a known enzyme which occurs in small amounts in the urine of mammals and accordingly also in human urine. Urokinase is an activator and serves to change plasminogen into plasmin. This enzyme in turn can dissolve fibrinous clots. Therefore urokinase preparations are valuable products in pharmaceutical medicine for the treatment, for example, of thromboembolisms.
• adsorption agents include for example calcium carbonate, barium sulfate, aluminum oxide, calcium phosphate, zinc hydroxide, activated carbon, hydrated aluminum silicates such as bentonite and kaolin, ion exchange silicates, molecular sieves as well as a number of other organic and inorganic materials.
• affinitive adsorption solid matrices which e.g. contain the following compounds immobilized superficially: epsilon-aminocaproic acid and p-aminoenzamidine on Sepharose®, antibody for urokinase on Sepharose, arginine on polyacrylamide resins, agmaline-epsiton-caproic acid on Sepharose®, trypsin inhibitor on agarose, lysin or argenine on agcerose, trypsin inhibitor on Sepharose, urokinase inhibitor from human placenta on Sepharose and similar combinations.
• the urokinase generally appears to be obtained in the form of two compounds, wherein the higher molecular weight compound has an average molecular weight of around 54,000 and the lower molecular weight compound has an average molecular weight of around 33,000.
• the process of the invention to be described in further detail below there is obtained a urokinase whose average molecular weight only varies around an average value of approximately 54,000.
• the basis for this improvement of the product quality cannot yet be clearly explained, it is being investigated at present whether the urokinase with the lower average molecular weight is a breakdown product of the higher molecular weight.
• the process of the invention for the production of pure urokinase of higher concentration and activity starting from a crude urokinase solution prepurified and concentrated by known process is generally characterized by this urokinase crude solution in a medium with a pH of ⁇ 6 being contacted with a porous solid matrix having a high specific surface area on which surface aprotinin is immobilized by means of covalent chemical bonds, that the carrier medium is separated and that subsequently the adsorbed urokinase is eluted by means of an eluting agent at pH ⁇ 4.5, whereupon there can be followed the known manufacturing methods for the production of various pharmaceutical preparations.
• a special, industrially very important illustrative form of the above described process is characterized by the mentioned urokinase crude solution in a medium at pH ⁇ 6 being separated affinity-chromatographically in a column over a porous solid matrix having a high specific surface area on which surface aprotinin is immobilized by means of covalent chemical bonds and that subsequently the adsorbed urokinase of this type is eluted by means of an eluting agent at pH ⁇ 4.5.
• the urokinase crude solution employed should have a concentration of 2,000 to 10,000 international units (IU) urokinase per ml of solution, advantageously such a one with 3,000 to 5,000 IU urokinase per ml of solution as well as a purity of 200 to 2,000 IU urokinase per mg of protein present, advantageously such a one having 300 to 1,000 IU urokinase per mg of protein present.
• IU international units
• porous solid matrix there can be used various resins, for example, those based on polysaccharides, cellulose, agarose such as Sepharose of the Pharmacin AB, Sweden, dextran as for example Sephadex® of the Pharmacin AB Sweden, or based on copolymer addition products based on polyacrylamides with agarose such as Sephacryl® of the Pharmacin AB Sweden, or Ultrogen® of LKB.
• bond facilitators or spaces for example are epsilon-aminocaproic acid, in the international literature frequently designated by EACA (6-aminohexanoic acid), divinyl sulfone, various bis oxieranes or other compounds.
• the solid matrix can, as frequently is the case with affinity-chromatography, be previously activated. For example, this can occur by means of BrCN or other known activating agents.
• the process of the invention is not suited for working up of crude urine, the concentration of urokinase in this solution is too low.
• the mentioned crude urokinese solution can be brought to a total salt concentration of 0.5 to 1 mole by addition of various salts, for example NaCl or KCl.
• the adsorption agent can be conditioned with a liquid medium at a pH of 6 to 9 and a total salt concentration of 0.5 to 1 mole.
• buffers there can be used the known phosphate buffer (e.g. monopotassium phosphate-disodium phosphate) or tris buffer, but others can also be employed.
• salts for the conditioning there can be added NaCl or KCl or other similar compounds.
• the laden solid matrix can be subjected to an intermediate rinsing.
• an intermediate rinsing for this purpose there can be used a liquid agent with a 0.5 to 1 molar NaCl solution. Above all this intermediate washing serves for the removal of undesired proteins from the solid matrix. Washing with the agent can be continued until the agent on leaving is optically inactive at a wave length of 280 nm, i.e. shows practically no concentration of protein.
• eluting agent there can be used a solution of 0.5 to 1 molar NaCl or KCl. Since the elution according to the invention must be carried out at a pH ⁇ 4.5, there can be added, e.g. solutions of the above mentioned salts and concentrations at a pH of 2 to 4.5. Such solutions can be established, e.g., by addition of acetic acid or HCl, but other compounds can also be used.
• elution aids such as aminoacids, e.g., aminocarboxylic acids such as lysine or arginine or others.
• the process can comprise, consist essentially of or consist of the steps set forth and the compositions can comprise, consist essentially of or consist of the materials set forth.
• the adsorbed urokinase was subsequently elutal with a solution of 0.5 molar NaCl, which was adjusted to a pH of 2.5 with HCl.
• the laden resin was subsequently rinsed with 3 liters of the mentioned buffer solution and afterwards with 2 liters of a 1.0 molar NaCl solution.
• the adsorbed urokenase was subsequently eluted by means of a solution of 1.0 molar NaCl which had been adjusted with acetic acid to a pH of 3.9.

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• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Genetics & Genomics (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biomedical Technology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Engineering & Computer Science (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Enzymes And Modification Thereof (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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Cited By (13)

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Citations (3)

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• 1978
  • 1978-06-30 CH CH719778A patent/CH643297A5/de not_active IP Right Cessation
• 1979
  • 1979-06-18 AR AR276951A patent/AR220202A1/es active
  • 1979-06-19 IL IL57596A patent/IL57596A/xx unknown
  • 1979-06-20 DE DE2924744A patent/DE2924744C2/de not_active Expired
  • 1979-06-25 IT IT23835/79A patent/IT1121914B/it active Protection Beyond IP Right Term
  • 1979-06-28 FR FR7916757A patent/FR2429837A1/fr active Granted
  • 1979-06-28 GB GB7922471A patent/GB2025977B/en not_active Expired
  • 1979-06-28 NL NLAANVRAGE7905045,A patent/NL185573C/xx not_active IP Right Cessation
  • 1979-06-29 JP JP54082534A patent/JPS5832591B2/ja not_active Expired
  • 1979-06-29 US US06/053,552 patent/US4259447A/en not_active Expired - Lifetime
• 1993
  • 1993-06-24 NL NL930096C patent/NL930096I1/nl unknown

Patent Citations (3)

Non-Patent Citations (4)

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